Association between Organochlorine Pesticides and Thyroid Tumors; an in-silico and in-vivo Case Control Study

Purpose: In this study, we aimed to investigate the role of organochlorine pesticides (OCPs), in the development of thyroid tumors. Method: Seven derived OCPs measured by gas chromatography (GC), and enzyme activities of acetylcholinesterase (AChE), superoxide dismutase3 (SOD3), catalase (CAT), glutathione peroxidase3 (GPx3) and paraoxonase1 (PON1) and also malondialdehyde (MDA), total antioxidant capacity (TAC), protein carbonyl (PC), and nitric oxide (NO), as oxidative stress (OS) biomarkers were assessed in the blood of 61 patients with papillary thyroid carcinoma (PTC), 70 patients with benign thyroid nodules (BTN), and 73 healthy individuals. Furthermore, all the studied enzymes were docked against the measured OCPs. Results: The results revealed that β-HCH, γ-HCH, 2,4 DDE, 4,4 DDE, 2,4-DDT, and 4,4-DDT levels along with MDA, NO and PC levels were elevated, while AChE, SOD3, GPx3, CAT, and PON1 activities and TAC levels were decreased in the PTC and BTN groups compared with the control group. Conclusion: The results of present study indicated that OCPs might play a major role in the development of thyroid tumors by disturbing the thyroid gland through several mechanisms including generation of OS. Importantly, in-silico analysis conrmed the in vivo ndings. activity in the patient groups with the control subject, the mean of PON1 arylesterase activity was signicantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signicantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signicantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signicantly increased compared to the healthy control (P < 0.001). The levels of MDA (µM/ml) of PTC and BTN were signicantly increased in comparison with healthy control (P < 0.001). The level of TAC (µM) of PTC and BTN were signicantly decreased compared to healthy control (P < 0.001), and BTN was signicant decrease in comparison from PTC group (P = 0.042). The levels of PC (nmol/mg protein) of PTC and BTN were signicantly increased in comparison with healthy control (P < 0.001). signicantly < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signicantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signicantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signicantly increased compared to the healthy control (P < 0.001). The levels of MDA (µM/ml) of PTC and BTN were signicantly increased in comparison with healthy control (P < 0.001). The level of TAC (µM) of PTC and BTN were signicantly decreased compared to healthy control (P < 0.001), and BTN was signicant decrease in comparison from PTC group (P = 0.042). The levels of PC (nmol/mg protein) of PTC and BTN were signicantly increased in comparison with healthy control (P < 0.001). the mean of PON1 arylesterase activity was signicantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signicantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signicantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signicantly increased compared to the healthy control (P < 0.001). The levels of MDA (µM/ml) of PTC and BTN were signicantly increased in comparison with healthy control (P < 0.001). The level of TAC (µM) of PTC and BTN were signicantly decreased compared to healthy control (P < 0.001), and BTN was signicant decrease in comparison from PTC group (P = 0.042). The levels of PC (nmol/mg protein) of PTC and BTN were signicantly increased in comparison with healthy control (P < 0.001). was signicantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signicantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signicantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signicantly increased compared to the healthy control (P < 0.001). The levels of MDA (µM/ml) of PTC and BTN were signicantly increased in comparison with healthy control (P < 0.001). The level of TAC (µM) of PTC and BTN were signicantly decreased compared to healthy control (P < 0.001), and BTN was signicant decrease in comparison from PTC group (P = 0.042). The levels of PC (nmol/mg protein) of PTC and BTN were signicantly increased in comparison with healthy control (P < 0.001). Data are expressed as means ± SEM and comparisons were made by using the one-way ANOVA. There were signicantly higher levels of α-, β- and γ-HCH, 2, 4-DDE, 2, 4-DDT and 4, 4-DDT pesticides in the PTC and BTN as compared to the control group (P = 0.016 for α-HCH in the BTN group, P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH, P < 0.001 for 2,4-DDE, 2,4-DDT and 4,4- DDT in the PTC and the BTN group, respectively). AChE activity was signicantly decreased in the patient groups compared to the control group (P < 0.001) and the BTN as compared to the PTC (P = 0.02). = 0.014), while there was a negative correlation between AChE and MDA (r=-0.180; P = 0.014), NO (r=-0.209; P = 0.004), and PC (r=-0.267; P < 0.001). In addition, PON1 had a signicant negative correlation with MDA (r=-0.499, P < 0.001), NO (r=-0.359; P < 0.001), and PC (r=-0.428; P < 0.001). There was a signicant positive correlation between PON1 and TAC (r = 0.384; P < 0.001), SOD3 (r = 0.275; P < 0.001), and GPx3 (r = 0.272; P < 0.001). MDA levels showed a signicant positive correlation with NO (r = 0.349; P < 0.001) and PC (r = 0.421; P < 0.001), and a negative correlation with TAC (r=-0.382; P < 0.001), SOD3 (r=-0.242; P = 0.001), and GPx3 (r=-0.197; P = 0.007). TAC showed a signicant positive relation to SOD3 (r = 0.263; P < 0.001) and GPx3 (r = 0.251; P = 0.001) and a signicant negative relation to NO (r=-0.361; P < 0.001) and PC (r=-0.383; P < 0.001). There was a positive correlation between NO and PC (r = 0.532; P < 0.001); moreover, there was a negative correlation between NO and SOD3 (r=-0.156; P = 0.033) as well as NO and GPx3 (r=-0.173; P = 0.018). With regard to the levels of oxidative and antioxidant biomarkers and AChE activity, no signicant difference was found between the two genders in the PTC BTN There positive correlation between CAT and AChE (r = 0.354; P < 0.001), PON1 (r = 0.442; P < 0.001), TAC (r = 0.395; P < 0.001), SOD3 (r = 0.223; P = 0.002), and GPx3 (r = 0.293; P < 0.001), while CAT had a negative correlation with MDA (r=-0.426, P < 0.001), NO (r=-0.312; P < 0.001), PC (r=-0.324; P < 0.001). In addition, we analyzed the correlation of educational levels and farming experience with the serum of OCPs. The results indicated a positive correlation between 2,4-DDE (r = 0.195; P = 0.01) and 4,4-DDT (r = 0.296; P < 0.001) with and a positive correlation between α-HCH (r = 0.206; P = 0.014), 2,4-DDE (r = 0.195; P = 0.02), 2,4-DDT (r = 0.201; P = 0.01), and 4,4-DDT (r = 0.240; P = 0.003) with farming experience. T3 had a negative correlation with 2,4-DDE (r=-0.207; P = 0.018). There was a positive correlation between T4 and γ-HCH (r = 0.161; P = 0.039), 2,4-DDT (r = 0.167; P = 0.033), and 4,4-DDT (r = 0.207; P = 0.007). TSH signicant P β-HCH (β=-0.149, P = 0.021), 2,4 DDE (β=-0.075, P = 0.003), 4,4-DDE (β=-0.15, P = 0.006), 2,4 DDT (β=-0.333, P = 0.008), and 4,4 DDT (β=-0.130, P = 0.001). lnPON1 activity had an inverse signicant association with α-HCH (β=-0.176, P = 0.012), β-HCH (β=-0.210, P = 0.003), γ-HCH (β=-0.23, P = 0.001), 2,4 DDE (β=-0.36, P < 0.001), 4,4-DDE (β=-0.20, P = 0.004), 2,4 DDT (β=-0.17, P = 0.015), and 4,4 DDT (β=-0.34, P < 0.001). SOD3 activity had a negative signicant association with 4,4 DDT (β=-0.91, P = 0.005) and a positive signicant association with α-HCH (β = 2.4, P = 0.001). GPx3 activity was signicantly related to 2,4 DDE (β=-0.41, P = 0.01), 2,4 DDT (β=-1.68, P = 0.03), and 4,4 DDT (β=-0.99, P < 0.001). lnCAT activity showed a signicant relationship with γ-HCH (β=-0.15, P = 0.02), 2,4 DDE (β=-0.23, P = 0.001), 4,4-DDE (β=-0.15, P = 0.029), 2,4 DDT (β=-0.29, P < 0.001), and 4,4 DDT (β=-0.32, P < 0.001). between and In study increased MDA levels and decreased SOD3 and GPx3 activities in both PTC and BTN and reduction in TAC in the patients with PTC compared to the control group. The reduction of TAC levels, SOD3 and GPx3 activities in thyroid cancer is thought caused by lipid peroxidation and damage to the antioxidant to pesticides. and Chol and the results demonstrated that these factors enhanced the odds of developing thyroid tumors. The decreased adjusted odds ratio of OCPs, such as 2,4-DDE, 4,4-DDE, and 2,4-DDT is due to the lower age of patients compared to the control group. The increase of OS and exposure to pesticides could predict the development of thyroid tumors with the AUC of 0.91. The ability to predict thyroid tumors by using the AUC of OCPs’ ROC curve was 0.72–0.92. Therefore, it can be concluded that OCPs enhance the levels of ROS related disease intensity.

for identifying OCPs residues as described elsewhere (10,11,22). First, the internal standard (4, 4-Dichlorobenzophenone (DBP)) was mixed with 0.5 ml of serum. The extraction of samples was repeated twice with 2 mL of hexane. Then, 200 µL of concentrated sulphuric acid was added to combined extracts in order to separate its organic part. Next, 100 mg of anhydrous sodium sulfate was used to dehydrate this organic part. Then, the transferred organic layer was completely concentrated at room temperature and in the next step, this concentrated organic layer was centrifuged. Eventually, 100 µL of Ethyl acetate was added to each sample in order to solve extracted OCPs. GC-FID and capillary columns (HP-5) are reported as analytical methods for the identi cation of OCPs (22). The retention time (that is a method of qualitative analysis), peak area (that is a method of quantitative analysis) and internal standard method were used to calculate the accumulation serum levels of OCPs. Therefore, a set of OCP standard solutions with determined concentrations (0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/ml) was prepared, and then equal values of DBP that is an internal standard (100 µg/ml) were added to each OCP standard solution. After injection to GC their gure of merits was obtained, and peak areas of OCPs standards and DBP were calculated. Finally, seven calibration curves have schemed for seven OCP compounds displaying the ratio of the peak area of OCP standard to internal standard versus concentration ratio. The areas of the OCPs and internal standard for unknown samples were calculated and the ratio of the peak area was reported. In the nal step, OCP standard curves were used to determine OCPs concentrations. The analytical limit of detection was estimated to be 0.7 µg/ml for α-HCH, β-HCH, γ-HCH, 2,4-DDE and 4,4-DDE and 3 µg/ml for 2,4-DDT, 4,4-DDT. We examined the quanti cation standard at the beginning and after the completion of a run. Moreover, the limit of detection (LOD) has been described as the concentration of compositions in the quanti cation standard divided by 3 times the ratio of signal-to-noise.

Molecular docking studies
In an in-silico analysis, AChE, SOD3, GPx3, CAT, and PON1 enzymes were docked against OCPs. The docking study was performed by Schrödinger-Glide with extra precision (XP) mode. The utilizing Protein Preparation Wizard of Schrodinger was prepared the crystal structure of AChE (Protein Data Bank (PDB) ID: 4PQE; resolution: 2.9 Å), PON1 (PDB ID: 1V04; resolution: 2.2 Å), CAT (PDB ID: 1DGB; resolution: 2.2 Å), GPx3 (PDB ID: 2R37; resolution: 1.85 Å), and SOD3 (PDB ID: 2JLP; resolution: 1.7 Å) by eliminating all the water molecules from crystal structure, and then the minimization was done with a cut off of 0.3 Å. The ligand le was acquired from the Zinc database. In order to the preparation of ligands was used LigPrep. The docking score, indicating values of the free energy of proteinligand binding, was attained using the Glide/XP docking.
2.14 Quality assurance and quality control (QA/QC) QA/QC was maintained to ensure the accurate quanti cation of OCPs. All the samples were analyzed in triplicate, and also eld blanks and equipment blanks were included. All the analytical results reported are the average of three values so that the method performance can be evaluated. In this regard, a set of pesticides' standards solutions with known concentrations (0.05, 0.1, 0.5, 0.75, 1,2,4,8,16,25,50, 100 µg/L) were spiked in the pooled sample, and the calibration curves were obtained. Procedure blanks were prepared using ethyl acetate and routinely analyzed to check for inlet, column, and detector contamination during extraction and injection, to examine the cross-contamination and to monitor the background contamination of the instrument.

Statistical analysis
Mean ± standard errors of the mean (mean ± SEM) were used to represent all continuous variable data and numbers (percentages) were used to represent categorical variables. Data distribution was determined using the Kolmogorov-Smirnov test. One-way Analysis of Variance (ANOVA)/Kruskal-Wallis with post-hoc Tukey/Mann-Whitney U tests, and Chisquare/Fisher's exact tests were used to show the differences among groups. Spearman' rho correlation coe cient was used to manifest the correlations between continuous variables. SPSS software version 22.0 for Windows (SPSS Inc., Chicago, IL) was used for the statistical analyses. In the present study linear regressions was carried out to nd out the effects of OCPs (as independent variables) on OS (as dependent variable) development. Six OS parameters showed approximately normal distributions, whereas TAC and PON-1 levels, and CAT activity were skewed distributions and were thus log-transformed to improve the normality assumption of the linear model. P values < 0.05 were considered as statistically signi cant. The associations between continuous OCP concentrations and OS parameters in all plasma subjects were explored using multivariable linear regression models. The estimation of the associations between thyroid cancer (PTC and BTN) development and OCPs was performed by the continuous logistic regression model, based on adjustments for age, smoking, hereditary, exposure, total lipids. Present study assessed exposures as categorical variables, by classifying each OCP as quartiles of exposure in the study population. For each OCP, we determined the OR for PTC comparing each quartile with quartile 1. All measurements for the studied pesticides were detected above the LOD. The measurement of LOD was based on the standard deviation of the regression line and slope of calibration curve. Also, we used both wet-weight concentrations adjusted for serum cholesterol and triglyceride and lipid-standardized concentrations by dividing wet-weight concentrations by total lipids. Total lipids were calculated using the short formula: total lipids (mg/dL) = 2.27 × total cholesterol + triglycerides + 62.3 (23).
In addition to individual kinds of OCP, we calculated molar sums (mmol/L) of DDT and its metabolites (2,4 DDT and 4,4 DDT), PHCHs (a-HCH, b-HCH, g-HCH,), and DDE (2,4 DDE and 4,4 DDE), using a reported method (24). The constructed interactions model was evaluated by receiver operating characteristics (ROC) analysis and area under the curve (AUC) calculations. ROC curves for models to predict the effect of each studied pesticides in thyroid cancer developments. Kaplan-Meier method was applied to estimate the survival functions of thyroid cancer patients submitted to surgery and grouped according to OCPs.

Demographic and clinical characteristics
Socio-demographics, lifestyle factors, and thyroid disease history of the study population are presented in Table 1. The mean age of the participants was 40 years (SEM ± 1.9) for the papillary thyroid carcinoma (PTC) group and 47 years for the benign thyroid nodules (BTN) group, which were signi cantly different between the two groups. There were 46 female subjects (75.4%) and 15 male subjects (24.6%) in the PTC group. In the BTN group, there were 56 female subjects (80%) and 14 male subjects (20%). Only 10.4% of the participants reported current smoking habits; 3.3% (2 out of 61) of the PTC and 5.7% (4 out of 70) of the BTN group were current smokers. A total of 23 individuals reported a family history of thyroid disease which includes 8 (13.1%) subjects in the PTC and 15 (21.4%) subjects in the BTN group. As observed, there were more subjects with a family history of thyroid disease in the BTN group than in the PTC group (P < 0.001). The levels of education and the levels of T3 and T4 were signi cantly higher in the patient groups compared to the control group.

Biochemical factors
There was no signi cant difference in the concentrations of total cholesterol, LDL-c, HDL-c, and triglycerides between the PTC and BTN groups compared to the healthy controls ( Table 1). The serum concentrations of T3 and T4 were signi cantly higher in the PTC and BTN groups as compared to the control group (P < 0.001, P = 0.007, respectively). The levels of TSH were signi cantly lower in the PTC and BTN groups compared to the healthy control group (P < 0.001).

Erythrocyte acetylcholinesterase activity
The mean activity of AChE are presented in Table 2. The measurement of AChE activity in the healthy and thyroid disease samples ( Figure S13 (a)) revealed a higher AChE activity in the healthy group in comparison with the BTN and PTC groups (P < 0.001). Evaluation of the AChE activity in the PTC and BTN groups con rmed that this enzyme's activity increased signi cantly in the PTC group compared to the BTN group (P = 0.0200). Data are expressed as means ± SEM and comparisons were made by using the one-way ANOVA. There were signi cantly higher levels of α-, β-and γ-HCH, 2, 4-DDE, 2, 4-DDT and 4, 4-DDT pesticides in the PTC and BTN as compared to the control group (P = 0.016 for α-HCH in the BTN group, P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH, P < 0.001 for 2,4-DDE, 2,4-DDT and 4,4-DDT in the PTC and the BTN group, respectively). AChE activity was signi cantly decreased in the patient groups compared to the control group (P < 0.001) and the BTN as compared to the PTC (P = 0.02). Comparison of PON1 arylesterase activity in the patient groups with the control subject, the mean of PON1 arylesterase activity was signi cantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signi cantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signi cantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signi cantly increased compared to the healthy control (P < 0.001). The levels of MDA (µM/ml) of PTC and BTN were signi cantly increased in comparison with healthy control (P < 0.001). The level of TAC (µM) of PTC and BTN were signi cantly decreased compared to healthy control (P < 0.001), and BTN was signi cant decrease in comparison from PTC group (P = 0.042). The levels of PC (nmol/mg protein) of PTC and BTN were signi cantly increased in comparison with healthy control (P < 0.001). Data are expressed as means ± SEM and comparisons were made by using the one-way ANOVA. There were signi cantly higher levels of α-, β-and γ-HCH, 2, 4-DDE, 2, 4-DDT and 4, 4-DDT pesticides in the PTC and BTN as compared to the control group (P = 0.016 for α-HCH in the BTN group, P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH, P < 0.001 for 2,4-DDE, 2,4-DDT and 4,4-DDT in the PTC and the BTN group, respectively). AChE activity was signi cantly decreased in the patient groups compared to the control group (P < 0.001) and the BTN as compared to the PTC (P = 0.02). Comparison of PON1 arylesterase activity in the patient groups with the control subject, the mean of PON1 arylesterase activity was signi cantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signi cantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signi cantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signi cantly increased compared to the healthy control (P < 0.001). The levels of MDA (µM/ml) of PTC and BTN were signi cantly increased in comparison with healthy control (P < 0.001). The level of TAC (µM) of PTC and BTN were signi cantly decreased compared to healthy control (P < 0.001), and BTN was signi cant decrease in comparison from PTC group (P = 0.042). The levels of PC (nmol/mg protein) of PTC and BTN were signi cantly increased in comparison with healthy control (P < 0.001). Data are expressed as means ± SEM and comparisons were made by using the one-way ANOVA. There were signi cantly higher levels of α-, β-and γ-HCH, 2, 4-DDE, 2, 4-DDT and 4, 4-DDT pesticides in the PTC and BTN as compared to the control group (P = 0.016 for α-HCH in the BTN group, P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH, P < 0.001 for 2,4-DDE, 2,4-DDT and 4,4-DDT in the PTC and the BTN group, respectively). AChE activity was signi cantly decreased in the patient groups compared to the control group (P < 0.001) and the BTN as compared to the PTC (P = 0.02). Comparison of PON1 arylesterase activity in the patient groups with the control subject, the mean of PON1 arylesterase activity was signi cantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signi cantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signi cantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signi cantly increased compared to the healthy control (P < 0.001). The levels of MDA (µM/ml) of PTC and BTN were signi cantly increased in comparison with healthy control (P < 0.001). The level of TAC (µM) of PTC and BTN were signi cantly decreased compared to healthy control (P < 0.001), and BTN was signi cant decrease in comparison from PTC group (P = 0.042). The levels of PC (nmol/mg protein) of PTC and BTN were signi cantly increased in comparison with healthy control (P < 0.001).  Data are expressed as means ± SEM and comparisons were made by using the one-way ANOVA. There were signi cantly higher levels of α-, β-and γ-HCH, 2, 4-DDE, 2, 4-DDT and 4, 4-DDT pesticides in the PTC and BTN as compared to the control group (P = 0.016 for α-HCH in the BTN group, P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH, P < 0.001 for 2,4-DDE, 2,4-DDT and 4,4-DDT in the PTC and the BTN group, respectively). AChE activity was signi cantly decreased in the patient groups compared to the control group (P < 0.001) and the BTN as compared to the PTC (P = 0.02). Comparison of PON1 arylesterase activity in the patient groups with the control subject, the mean of PON1 arylesterase activity was signi cantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signi cantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signi cantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signi cantly increased compared to the healthy control (P < 0.001). Data are expressed as means ± SEM and comparisons were made by using the one-way ANOVA. There were signi cantly higher levels of α-, β-and γ-HCH, 2, 4-DDE, 2, 4-DDT and 4, 4-DDT pesticides in the PTC and BTN as compared to the control group (P = 0.016 for α-HCH in the BTN group, P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH, P < 0.001 for 2,4-DDE, 2,4-DDT and 4,4-DDT in the PTC and the BTN group, respectively). AChE activity was signi cantly decreased in the patient groups compared to the control group (P < 0.001) and the BTN as compared to the PTC (P = 0.02). Comparison of PON1 arylesterase activity in the patient groups with the control subject, the mean of PON1 arylesterase activity was signi cantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signi cantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signi cantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signi cantly increased compared to the healthy control (P < 0.001). Data are expressed as means ± SEM and comparisons were made by using the one-way ANOVA. There were signi cantly higher levels of α-, β-and γ-HCH, 2, 4-DDE, 2, 4-DDT and 4, 4-DDT pesticides in the PTC and BTN as compared to the control group (P = 0.016 for α-HCH in the BTN group, P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH, P < 0.001 for 2,4-DDE, 2,4-DDT and 4,4-DDT in the PTC and the BTN group, respectively). AChE activity was signi cantly decreased in the patient groups compared to the control group (P < 0.001) and the BTN as compared to the PTC (P = 0.02). Comparison of PON1 arylesterase activity in the patient groups with the control subject, the mean of PON1 arylesterase activity was signi cantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signi cantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signi cantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signi cantly increased compared to the healthy control (P < 0.001). Data are expressed as means ± SEM and comparisons were made by using the one-way ANOVA. There were signi cantly higher levels of α-, β-and γ-HCH, 2, 4-DDE, 2, 4-DDT and 4, 4-DDT pesticides in the PTC and BTN as compared to the control group (P = 0.016 for α-HCH in the BTN group, P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH, P < 0.001 for 2,4-DDE, 2,4-DDT and 4,4-DDT in the PTC and the BTN group, respectively). AChE activity was signi cantly decreased in the patient groups compared to the control group (P < 0.001) and the BTN as compared to the PTC (P = 0.02). Comparison of PON1 arylesterase activity in the patient groups with the control subject, the mean of PON1 arylesterase activity was signi cantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signi cantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signi cantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signi cantly increased compared to the healthy control (P < 0.001).  Data are expressed as means ± SEM and comparisons were made by using the one-way ANOVA. There were signi cantly higher levels of α-, β-and γ-HCH, 2, 4-DDE, 2, 4-DDT and 4, 4-DDT pesticides in the PTC and BTN as compared to the control group (P = 0.016 for α-HCH in the BTN group, P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH, P < 0.001 for 2,4-DDE, 2,4-DDT and 4,4-DDT in the PTC and the BTN group, respectively). AChE activity was signi cantly decreased in the patient groups compared to the control group (P < 0.001) and the BTN as compared to the PTC (P = 0.02). Comparison of PON1 arylesterase activity in the patient groups with the control subject, the mean of PON1 arylesterase activity was signi cantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signi cantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signi cantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signi cantly increased compared to the healthy control (P < 0.001). The levels of MDA (µM/ml) of PTC and BTN were signi cantly increased in comparison with healthy control (P < 0.001). The level of TAC (µM) of PTC and BTN were signi cantly decreased compared to healthy control (P < 0.001), and BTN was signi cant decrease in comparison from PTC group (P = 0.042). The levels of PC (nmol/mg protein) of PTC and BTN were signi cantly increased in comparison with healthy control (P < 0.001).  Data are expressed as means ± SEM and comparisons were made by using the one-way ANOVA. There were signi cantly higher levels of α-, β-and γ-HCH, 2, 4-DDE, 2, 4-DDT and 4, 4-DDT pesticides in the PTC and BTN as compared to the control group (P = 0.016 for α-HCH in the BTN group, P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH, P < 0.001 for 2,4-DDE, 2,4-DDT and 4,4-DDT in the PTC and the BTN group, respectively). AChE activity was signi cantly decreased in the patient groups compared to the control group (P < 0.001) and the BTN as compared to the PTC (P = 0.02). Comparison of PON1 arylesterase activity in the patient groups with the control subject, the mean of PON1 arylesterase activity was signi cantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signi cantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signi cantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signi cantly increased compared to the healthy control (P < 0.001).  Data are expressed as means ± SEM and comparisons were made by using the one-way ANOVA. There were signi cantly higher levels of α-, β-and γ-HCH, 2, 4-DDE, 2, 4-DDT and 4, 4-DDT pesticides in the PTC and BTN as compared to the control group (P = 0.016 for α-HCH in the BTN group, P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH, P < 0.001 for 2,4-DDE, 2,4-DDT and 4,4-DDT in the PTC and the BTN group, respectively). AChE activity was signi cantly decreased in the patient groups compared to the control group (P < 0.001) and the BTN as compared to the PTC (P = 0.02). Comparison of PON1 arylesterase activity in the patient groups with the control subject, the mean of PON1 arylesterase activity was signi cantly decreased (P < 0.001). With respect to the SOD3 bar chart, the SOD3 activity in the patient groups have signi cantly decreased compared to the control group (P < 0.001). The mean of GPx3 activity decreased in the PTC and BTN groups in comparison with control ones (P < 0.001). CAT activity was signi cantly lower in the patient groups rather than the control group (P < 0.001). The level of NO (µmol/L) of PTC and BTN were signi cantly increased compared to the healthy control (P < 0.001). The levels of MDA (µM/ml) of PTC and BTN were signi cantly increased in comparison with healthy control (P < 0.001). The level of TAC (µM) of PTC and BTN were signi cantly decreased compared to healthy control (P < 0.001), and BTN was signi cant decrease in comparison from PTC group (P = 0.042). The levels of PC (nmol/mg protein) of PTC and BTN were signi cantly increased in comparison with healthy control (P < 0.001).

Oxidative stress (OS) parameters
The mean levels of oxidative and antioxidant biomarkers are presented in Table 2. Serum PON1 activity was signi cantly lower in the PTC and BTN groups compared to the healthy controls (P < 0.001). No signi cant differences were observed between the PTC and BTN groups in terms of PON1 activity (P = 0.9130) ( Figure S13 (b)). In the PTC group, SOD3 activity was not signi cantly different (P = 0.177) compared to the BTN group but was signi cantly decreased compared to the controls (P < 0.001) ( Figure S13 (c)). In the PTC group, GPx3 activity was signi cantly lower than the control group (P < 0.001), but was not signi cantly different (P = 0.387) compared to the BTN group ( Figure S13 (d)). Serum CAT activity was signi cantly lower in the PTC and BTN groups rather than the control group (P < 0.001).
There was no signi cant difference between the PTC and BTN groups in terms of CAT activity (P = 0.7550) ( Figure S13 (e)). The results also revealed that MDA increased in the PTC and BTN groups as compared to the control group (P < 0.001). However, the PTC and BTN groups showed no signi cant difference in the MDA levels (P = 0.547) ( Figure S13 (f)). Signi cant elevation of serum NO was found in the PTC and BTN groups as compared to the control group (P < 0.001). However, there was no signi cant difference between the PTC and BTN groups in terms of NO levels (P = 0.9590) ( Figure S13 (g)). TAC of the normal group was signi cantly higher than that of the PTC and BTN groups (P < 0.001). In addition, TAC was signi cantly different between the PTC and BTN groups (P = 0.0420) ( Figure S13 (h)). the healthy controls. There was no signi cant difference in PC levels between the PTC and BTN groups (P = 0.207) ( Figure S13 (i)).
Logistic regression analysis showed that higher levels of OCPs were associated with the risk of thyroid tumor (  The ability of OCPs to differentiate thyroid tumor patients from healthy individuals was evaluated using the ROC curve analysis. When using a 4,4-DDT cut-off of > 0.01500, the ROC curve had the highest AUC of 0.92, with a 95% con dence interval (CI) ranging from 0.88 to 0.95, a sensitivity of 95.42% and speci city of 84.73%. Following that was the ROC curve of 2,4-DDE with a cut-off of > 1.010, which had an AUC of 0.91 with a 95% CI ranging from 0.87 to 0.95, a sensitivity of 86.26% and speci city of 86.30%. The predictive accuracy expressed as the ROC-AUC increased slightly when exposure to pesticides was used along with OS; AUC ranged from 0.87 to 0.91 with a 95% CI ranging from 0.82 to 0.92 for OS and a 95% CI ranging from 0.88 to 0.93 for OS along with exposure to pesticides (Fig. 1).
To understand the interactions between the OCPs and antioxidant enzymes, a docking study was performed (Fig. 2, Table 4, and Figure  S1-5). Docking identi ed two binding sites for α, β, and γ-HCH in AChE, however, only one binding site was detected for 2,4-DDT and 4,4-DDE. Meanwhile, 4,4-DDT and 2,4-DDE were unable to interact with AChE. The α-and β-HCH and DDT and its derivatives inhibited PON1 near its active site and in the presence of PO 4 3− , while γ-HCH did not inhibit PON1. α, β and γ-HCH did not inhibit GPx3, but DDT and its derivatives inhibited this enzyme. All the studied OCPs inhibited SOD3 and CAT. Moreover, 2,4-DDT exhibited the lowest binding free energy (∆G) among all the OCPs (-86.10 kcal/mol).  As shown in Fig. 3, exposure to high amounts of 4,4-DDT was associated with a reduced survival rate in the PTC group (Hazard Ratio, 1.155; 95% CI, 1.011-1.320), however, other OCPs did not have a signi cant association with the survival rate (Fig. 3). The patients with PTC who were exposed to higher 4,4-DDT levels (> 7.5 ng/ml) were found to be at a signi cantly greater risk of PTC death (P = 0.03).

Organochlorine pesticides
Comparisons of the mean serum levels of OCPs in PTC, BTN, and control groups are presented in Table 2 and Figure S9 (c). Figures S9 (a) and S9 (b) depict the OCP chromatogram curve of the BTN and PTC patients, respectively. The levels of β-and γ-HCH, 2,4-DDE, 4,4-DDE, 2,4-DDT, and 4,4-DDT were signi cantly higher in the PTC and BTN groups as compared to the controls (P = 0.015 and P < 0.001 for β-HCH, P < 0.001 and P = 0.003 for γ-HCH in the PTC and the BTN groups, respectively; P < 0.001 for 2,4-DDE, 4,4-DDE, 2,4-DDT, and 4,4-DDT in both groups). The levels of α-HCH were signi cantly higher only in the BTN group as compared to the control group (P = 0.016). No signi cant difference was observed between the two genders in the PTC and BTN groups.
The heatmap plot and spearman correlation analysis showed a signi cant correlation between most of the studied parameters as depicted in Fig. 4 and Table S1. There was a signi cant positive association between the average OCP residual levels and MDA, NO and PC; however, signi cant negative correlations were found between the average OCP residual levels and PON1, AChE, SOD3, GPx3 and CAT activities and TAC levels. In addition, there was a direct relation between the thyroid tumors progression and a more severe stage/histopathology with higher exposure to OCPs ( Figure S7, S8, and Table S1).
There  (Table S2). As shown in Table S2, after adjusting for confounding factors such as age, smoking, exposure to pesticides, and total lipids decreased OCPs effects on OS biomarkers, except for 4,4-DDT on NO, which was strengthened by these confounders. Furthermore, Fig. 5 was presented to demonstrate the associations between quartiles of ∑OCPs levels and OS biomarker levels. We found that in the 2nd, 3rd, and 4th quartile of ∑OCPs had a signi cant decrease of AChE, and in the 4th quartile of ∑OCPs had a signi cant decrease of GPx3, lnCAT, lnTAC, levels and lnPON1. Also, in the 2nd, 3rd, and 4th quartile of ∑OCPs had a signi cant increase of MDA, and in the 3rd and 4th quartile of ∑OCPs had a signi cant increase of NO and PC. The associations between quartiles of ∑HCH, ∑DDE, and ∑DDT levels and OS biomarker levels were shown in Figure S10-S12.

Discussion
The present study showed that β-HCH, γ-HCH, 2, 4 DDE, 4,4-DDE, 2, 4-DDT and 4, 4-DDT had signi cantly higher levels in the PTC and BTN groups as compared to the control group. Therefore, it can be concluded that these pesticides are risk factors contributing to the development of thyroid tumors in the studied area ( Figure S6). In the current study, the mean age of participants was 40 years in the PTC group, and the prevalence of the PTC was higher in women. The family history, HDL-c, LDL-c, cholesterol, and triglyceride levels were not associated with an increased risk of PTC. Educational levels had a positive association with the exposure to 2,4-DDE and 2,4-DDT, as presented in Table S1. Moreover, increased levels of MDA, NO, PC and decreased AChE, SOD3, GPx3, PON1 and CAT activities and TAC levels in the PTC and BTN groups were observed when compared with the normal healthy group. Furthermore, logistic regression analyses revealed an association between OCPs and disease status, and linear regression analysis indicated a signi cant relationship between the levels of OS biomarkers and OCP exposure and attenuation of OCPs effect on OS could due to lower age and smoking patients than healthy group and protective effect of these factors. The docking results veri ed our experimental ndings. In addition, using ROC analysis, we con rmed that OCP exposure levels could be a predictor of the development of thyroid tumors. Investigating the survival rate showed that high exposure to 4,4-DDT increases mortality in PTC patients.
Moreover, this research suggested that the risk of the development of thyroid cancer among women who are exposed to pesticides is higher compared to men. This nding is supported by a previous study that reported among all the individuals who are exposed to pesticides, women are more likely to develop thyroid cancer compared to men (25). This difference is especially noticeable for PTC in comparison with other main histological subtypes of thyroid cancer (26).
Age, as a unique hallmark of cancer, predicts the well-differentiated thyroid cancer in a prognostic manner. It is seen that patients aged over 45 years run the same risk of disease involvement but have a very different prognosis compared to those aged less than 45 years.
The reason, however, for why age is associated with the outcome is not fully understood (27). It is noteworthy that these ndings were not consistent with those of our study. These differences may be attributed to various reasons such as eating habits, ethnicity, race, socioeconomic status, and environmental and occupational factors.
The risk of developing PTC in women who had a history of thyroid disease in their immediate family was not signi cant, thereby suggesting that a family history of thyroid cancer does not play a prominent role in cancer incidence. History of thyroid cancer among immediate family members, particularly siblings, is reported to have a direct association with an increased risk of sporadic PTC (28).
Current results showed more exposed to 2,4-DDE and 2,4-DDT was associated with higher education levels. Junque et al. observed a positive correlation between social class and education level with 4,4-DDE levels in maternal venous blood collected in the rst trimester and cord blood (29). This nding is acceptable because higher education can increase knowledge and lead to a desire for healthy diet includes fruits, vegetables. Using these nutritional values, which are more probably exposed to pesticides and maintain them in themselves, pesticides reach the human body through these pathways. We found that the levels of HDL-c, LDL-c, cholesterol and triglyceride were not signi cantly different in the control and PTC subjects.
Similar results have been observed in a study conducted by Giusti (30) where no lipid pro le changes were seen in the differentiated thyroid carcinoma (DTC) patients. Additionally, the results showed that there was no relationship between serum concentrations of lipids and OCPs. As shown in Table 1, the levels of T3 and T4 were signi cantly higher in the patient groups compared to the control group whereas a signi cant reduction of serum TSH was found in the PTC and BTN groups as compared to the control group. This shows the feedback effect of T3 and T4 on hypothalamus and hypothesis in the PTC and BTN patients as anticipated. Consistently, Blanco-Muñoz et al. reported positive associations between the serum levels of p,p'-DDE and those of total T3, and total T4 and negative but no signi cant changes in TSH in male farmworkers (8).
We showed that there was a negative correlation between OCPs and AChE, PON1, SOD3, GPx3, and CAT activities and TAC levels.
Consistently, a previous study reported a relationship between pesticide exposure and the decrease of AChE activity with antioxidant enzymes (SOD3 and CAT), however, in their study GPx3 activity did not change (31). Also, our results showed that there was a positive correlation between OCPs and MDA, NO and PC. In our study increased MDA levels and decreased SOD3 and GPx3 activities were found in both PTC and BTN patients and reduction in TAC in the patients with PTC compared to the control group. The reduction of TAC levels, SOD3 and GPx3 activities in thyroid cancer is thought to be caused by the increased lipid peroxidation and damage to the antioxidant defense system due to increased exposure to pesticides.
The scienti c literature abounds in the studies that have examined AChE activity in a wide range of human diseases; however, to the best of our knowledge, this is the rst report on the activity of AChE in PTC and BTN patients. A study on the AChE activity in colorectal cancer (CRC) patients showed a signi cant reduction of serum erythrocyte AChE activity in the CRC patients that exposure to pesticides compared to the control subjects (10). In our study, it was found that AChE activity was lower in the PTC and BTN patients compared to the control group; moreover, AChE activity in the BTN group signi cantly decreased as compared to the PTC group. In fact, there is a signi cant correlation between the reduction of AChE activity and the reduction of TAC levels. In addition, the results manifested that the TAC levels and AChE activity were higher in the PTC group compared to the BTN group, indicating that the levels of OS were higher in the PTC group compared to the BTN group. It can be concluded that the OS levels depend on the type of pesticides as well as the amount and period of exposure, as reported previously (9)(10)(11)32).
PON1 activity had a decreasing trend in patients with PTC and a signi cant increasing trend after total thyroidectomy (33). Consistently, in the current study, we observed lower serum PON1 activity in the PTC and BTN patients compared to the healthy subjects. It was also shown that oxidized lipids contribute to the inhibition of PON activity (33). Our assumption was that increased levels of MDA might be one of the reasons why PON1 activity was reduced in our study. Furthermore, we found a positive correlation between serum PON1 and AChE activities in the participants exposed to pesticides. This result was in line with the results of a previous investigation (34,35).
Therefore, it could be concluded OCPs through inhibition of PON1 and AChE, and probably other antioxidant enzymes, increase OS and by this way contribute to the development of thyroid tumors.
It is well grounded that NO affects the iodine thyroid metabolism as nitrate competitively inhibits sodium/iodide cotransporter (also dubbed symporter) and prevents iodide uptake by the gland; as a consequence, thyroid hormone synthesis is compromised, which leads to the thyrotropin elevation. Therefore, chronic thyroid stimulation, due to pesticides exposure, can lead to proliferative changes e.g.
hypertrophy and hyperplasia as well as neoplasia as described by other researchers too (36). The results of the present study indicated that the increased NO levels were also more evident in the PTC group subjects who were highly exposed to OCPs (β-HCH, γ-HCH, 2,4-DDE, 4,4-DDE, and 4,4-DDT) compared to the control group. These ndings supported the results of a previous study that reported the levels of NO correlated with the progression and metastasis of PTC (37).
The presence of PC is considered as a biomarker of ROS-mediated protein oxidation (38). It is reported that MDA can irreversibly bind to proteins via covalent bonds, thereby increasing the formation of PC (39). In accordance with these ndings, we witnessed a signi cant correlation between MDA and PC in the PTC group. These observations were absent in the control group which may be due to the lower OS levels in this group. Furthermore, logistic regression analysis was performed by adjusting for age, smoking, heredity, exposure to the pesticides, TG, and Chol and the results demonstrated that these factors enhanced the odds of developing thyroid tumors. The decreased adjusted odds ratio of OCPs, such as 2,4-DDE, 4,4-DDE, and 2,4-DDT is due to the lower age of patients compared to the control group.
The increase of OS and exposure to pesticides could predict the development of thyroid tumors with the AUC of 0.91. The ability to predict thyroid tumors by using the AUC of OCPs' ROC curve was 0.72-0.92. Therefore, it can be concluded that OCPs enhance the levels of ROS related disease intensity.
An important part of the present study was the in-silico analysis of AChE, SOD3, GPx3, CAT, and PON1 enzymes with the OCPs. Interestingly, the results of modeling these molecules supported the in-vivo experiments. Between all OCPs, 2,4-DDT presented the highest binding with SOD3 and CAT (∆G of -86.10 kcal/mol and − 84.13 kcal/mol, respectively). Therefore, it can be concluded that 2,4-DDT has the highest levels of toxicity in the body. However Interestingly, the OCPs bonded to another site of SOD3 and GPx3 instead of their active site. The OCPs' binding modes showed that these pesticides changed protein conformations and inhibited the activities of PON1, CAT, SOD3, and GPx3 ( Figure S1-5).
Also, the present study has shown that high exposure to 4,4-DDT is associated with increased mortality for PTC. In Parada et al. study, 4,4-DDT was adversely associated with survival in women with breast cancer (41), which is consistent with the results reported here.

Conclusion
The ndings of the present study indicated that OCPs might play an important role in the thyroid tumor incidence and exert disturbance in the cells through several mechanisms including OS, as we have successfully shown that OCPs affect important enzymes involved in oxidoreductase reactions in an in-silico analysis. In conclusion, accumulation data obtained from patients with thyroid tumors and insilico analysis suggest that OCPs play an important role in the development of thyroid tumors in southeast Iran.